The overall objective of this program project grant is to develop and test genetically engineered herpes simplex viruses (HSV) that can be used safely and effectively to treat humans with malignant primary brain tumors. The goal of this project is to construct a series of recombinant HSV which exhibit a high level of selectivity for malignant glioma cells and that will produce a direct oncolytic effect or will induce oncolysis directly. We propose to provide at least 5 novel genetically engineered HSV annually for assessment of oncolytic activity in vitro and in relevant glioma models in experimental animal systems. In order to achieve this effectively and efficiently, we will develop a method for rapid construction of genetically engineered HSV based on cosmids. We will determine the minimal the minimal packaging capacity of the HSV genome and will construct vectors capable of carrying multiple non-viral genes. Finally, to provide a margin of safety and to better control the timing and extent of the oncolytic effect, we will seek to create conditions under which expression of specific genes can be controlled in vivo using specific promoters. It is a goal to be able to construct attenuated genetically engineered HSV suitable for intracerebral inoculation into a wide variety of experimental animal systems (including simian primate species of greater susceptibility than humans) and that exhibit the potential to discriminate between normal and malignant cells. As a means of amplifying the oncolytic effect, we will construct genetically engineered HSV that carry foreign genes, the products of which will produce a cytotoxic effect directly or will act to induce a radiation sensitizing effect or to elicit an anti-tumor immune-related inflammatory response.